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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD 471, AMES: Negative 

OECD 473, In vitro Chromosome Aberration in v79 cells: Negative

OECD 476, In vitro Gene Mutation HPRT : Negative

OECD 474, in vivo mouse micronuclues: Negative

Link to relevant study records

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002-09-11 to 2002-10-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Principles of method if other than guideline:
Note: Both direct plate assay and preincubation assay methods applied.
GLP compliance:
yes
Remarks:
No certificate
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): Lemonile
- Substance type: Colourless to pale yellow liquid
- Physical state: Liquid
- Analytical purity: 99.4 %
- Impurities (identity and concentrations): No data
- Composition of test material, percentage of components: No data
- Isomers composition: No data
- Purity test date: No data
- Batch No: 9000477843
- Expiration date of the batch: 2004-07-11
- Stability under test conditions (in vehicle DMSO): "not indicated"
- Storage condition of test material: At room temperature in the dark
- Stability under storage conditions: Stable
- Specific gravity: 0.8647
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
other: See text
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Dose range-finding test 1 (direct plate assay): 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate.
Dose range-finding test 2 (preincubation assay): 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate.
Direct plate assay 1: 3, 10, 33, 100, 333, 1000, 3330 and 666 µg/plate.
Direct plate assay 2: 100, 333, 1000 and 3330 µg/plate.
Preincubation assay 1: 10, 33, 100, 333, 1000 and 2000 µg/plate.
Preincubation assay 2: 1, 3, 10, 33, 100, and 333 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: No data
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
Without metabolic activation. Strain - TA1535. Solvent - saline. Concentration/plate - 5 µg.
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without metabolic activation. Strain - TA1537. Solvent - saline. Concentration/plate - 60 µg. Direct plate assay only.
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Without metabolic activation. Strain - TA1537. Solvent - saline. Concentration/plate - 15 µg. Preincubation assay only.
Positive controls:
yes
Positive control substance:
other: Daunorubicin
Remarks:
Without metabolic activation. Strain - TA98. Solvent - saline. Concentration/plate - 4 µg.
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without metabolic activation. Strain - TA100. Solvent - DMSO. Concentration/plate - 650 µg.
Positive controls:
yes
Positive control substance:
cumene hydroperoxide
Remarks:
Without metabolic activation. Strain - TA102. Solvent - DMSO. Concentration/plate - 0.1 µg.
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene [EC name: 2-anthrylamine]
Remarks:
With metabolic activation. Strains - TA1535, TA1537, TA98, TA100. Solvent - DMSO. Concentrations/plate - 1 µg (TA1535, TA98, TA100), 2.5 µg (TA1537).
Positive controls:
yes
Positive control substance:
other: 1,8-Dihydroxyanthraquinone [EC name: dantron]
Remarks:
With metabolic activation. Strain - TA102. Solvent - DMSO. Concentration/plate - 30 µg.
Details on test system and experimental conditions:
METHODS OF APPLICATION: In agar (plate incorporation); preincubation

STRAINS
- Strains were obtained from Dr Bruce N. Ames, University of California at Berkeley, United States. For details on the strains used, see the "Any other information materials and methods incl. tables" section below.
- Strains were regularly checked to confirm their histidine requirement, crystal violet sensitivity, ampicillin resistance (TA98, TA100 and TA102), tetracycline resistance (TA102), UV-sensitivity and the number of spontaneous revertants.
- Each tester strain contained the following additional mutations:
-- rfa deep rough (defective lipopolysaccharide cellcoat)
-- gal mutation in the galactose metabolism
-- chl mutation in nitrate reductase
-- bio defective biotin synthesis
-- uvrB loss of the excision repair system (deletion of the ultraviolet repair B gene)
- Stock cultures of tester strains were stored in liquid nitrogen (-196 °C)

CELL CULTURE
- Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid No. 2) and incubated in a shaking incubator (37 °C, 150 spm) until the cultures reached an optical density of 1 ± 0.1 at 700 nm (10⁹ cells/mL). Freshly grown cultures of each strain were used for a test.
- Agar plates (9 cm diameter) contained 25 mL glucose agar medium. Glucose agar medium contained, per litre, 18 g purified agar (oxoid code L28) in Vogel-Bonner medium E, 20 g glucose, 0.5 mg biotin and 0.6 mg histidine
- Top agar medium contained 0.6 % (w/v) agar and 0.5 % (w/v) NaCl, heated to dissolve the agar. Samples of 3 mL top agar were transferred into 10 mL glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 1 °C.
- Incubations performed in dark at 37 ± 1 °C. Temperature monitored.

METABOLIC ACTIVATION SYSTEM
Preparation of S9 Fraction:
- Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River, Sulzfeld, Germany
- Animals housed at NOTOX in a special room under standard laboratory conditions, as described in the SOP.
- The rats were injected intraperitoneally with a solution of 20% (w/v) Aroclor 1254 (500 mg/kg bw) in corn oil. 5 days later, the rats were sacrificed by decapitation, having been denied food for at least 12 hrs.
- The livers of the rats were removed aseptically and washed in cold (0 °C) sterile 0.1 M sodium phosphate buffer (pH 7.1) containing 0.1 mM Na₂-EDTA. Subsequently the livers were minced in a blender and homogenised in 3 volumes of phosphate buffer with a Potter homogeniser. The homogenate was centrifuged for 15 mins at 9000 G. The supernatant (S9) was transferred into sterile ampules, which were stored in liquid nitrogen (-196 °C) and identified by the day of preparation.
- Before use, all S9-batches were characterised with the metabolic activation requiring positive control: benzo[a]pyrene (Sigma)

Preparation of S9-Mix:
- S9 mix was prepared immediately before use and kept on ice. S9-mix contained, per 10 mL, 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 mL Milli-Q water, 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl₂ solution, 1 mL of 0.33 M KCl solution.
- The above solution was filtered (0.22 µm)-sterilised. To 9.5 mL of S9-mix components, 0.5 mL S9-fraction (batches 0-26 and 0-27) was added 5 % (v/v) S9-fraction to complete the S9-mix.

TEST PROCEDURE
Dose range-finding tests:
- Dose range-finding tests were performed to both methods (direct plate and preincubation)
- Selection of a range of doses was based on dose-range finding tests both with and without S9-mix. 8 concentrations were tested in triplicate, and the results formed part of the complete dataset.
- The highest concentration of test material used in the subsequent mutation assay was the level at which the test substance inhibited bacterial growth or the test substance exhibited limited solubility.

Mutation assay:
- ≥ 5 different doses, at approx. half-log steps, were tested in triplicate at each strain.
- Test substance was tested both with and without S9-mix.
- 2 additional experiments were performed with TA1535, TA1537, T98 and TA102; one direct plate assay and one preincubation assay.

Direct plate assay:
- Top agar in top agar tubes was molten and heated to 45 °C. The following solutions were successively added to 3 mL molten agar: 1 mL of a fresh bacterial culture 10⁹ cells/mL of one of the tester strains; 0.1 mL of a dilution of the test substance in DMSO; and either 0.5 mL S9-mix or 0.5 mL 0.1 M phosphate buffer (as appropriate). The ingredients were mixed on a Vortex and the contents of the top agar tube were poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark at 37 °C for 48 hr. After this period the revertant colonies were counted.

Preincubation assay:
- Top agar in top agar tubes was molten and heated to 45 °C. The following solutions were preincubated for 30 mins by 70 rpm at 37 °C, either 0.5 mL S9-mix or 0.1 M phosphate buffer (as appropriate), 0.1 mL of a fresh bacterial culture (10⁹ cells/mL) of one of the tester strains, 0.1 mL of test material in DMSO.
- After preincubation period the solutions were added to 3 mL molten top agar. The ingredients were mixed using a vortex and the contents of the top agar tube were poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark at 37 °C for 48 hr. After this period the revertant colonies were counted.

Evaluation
- Revertant colonies automatically counted using a Protos model 50000 colony counter or manually if fewer than 40 colonies per plate were present. Plates with sufficient test article precipitate to interfere with automatic colony counting were counted manually.
- Further details on evaluation are included in Appendix 1 (attached).
Evaluation criteria:
A substance is considered negative (not mutagenic) in the test if:
1. The number of revertants in any tester strain at any concentration is not greater than 2 times the solvent control value, with or without metabolic activation.
2. The negative response should be reproducible in at least one repeat experiment.

A test substance was considered positive (mutagenic) in the test if:
1. It induces at least a twofold dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, with or without metabolic activation. However, any plate count of less than 20 is considered not to be significant.
2. The positive response should be reproducible in at least one repeated experiment.

The above criteria were not absolute and other modifying factors could enter into the final evaluation decision.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
≥333 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA:
- Historical control data are presented in the additional materials attached.
- The negative and strain-specific positive values were within laboratory background historical control data ranges, indicating that the test conditions were adequate and the metabolic activation system functioned properly.
Remarks on result:
other: all strains/cell types tested

For results tables, please see the attached supporting information.

Conclusions:
The test material was found not to be mutagenic in an Ames test with Salmonella typhimurium strains TA100, TA1535, TA1537, TA98 and TA102.
Executive summary:

Lemonile was tested in the Salmonella typhimurium reverse mutation assay with the five histidine-requiring strains of Salmonella typhimurium (TA 1535, TA1537, TA98, TA100 and TA102). The test was performed in two separate experiments in the presence and absence of S9 -mix (Aroclor-1254 induced rat liver S9 -mix). To obtain more information about the mutagenicity of Lemonile, two additional experiments were performed with the strains TA1535, TA1537, TA98 and TA102; one direct plate assay and one preincubation assay.

In the direct plate assay, at first Lemonile was tested in a dose range finding study up to concentrations of 5000ug/plate in strain TA100. Lemonile was precipitated on the plates at dose levels of 3330 and 5000ug/plate. Toxicity was observed at dose levels of 333 ug/plate and upwards.

Secondly, Lemonile was tested up to concentrations of 666 ug/plate in the strains TA 1535, TA1537, TA98 and TA102. Lemonile did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested. A decrease in the number of revertants was observed in tester strain TA1535 in the absence and presence of S9 -mix and TA98 in the absence of S9 -mix.

In an additional direct plate assay, at first Lemonile was tested up to concentrations of 3330 ug/plate in the stains TA1535, TA1537, TA98 and TA102. Lemonile precipitated on the plates at this dose level. Toxicity was observed in these strains.

In the preincubation assay, at first Lemonile was tested in a dose range finding study in the strain TA100. Lemonile was tested up to concentrations of 5000 ug/plate. Lemonile precipitated on the plates at dose levels of 3330 and 5000 ug/plate. Toxicity was observed at dose levels of 33 ug/plate andupwards in the absence of S9 -mix and at 100 ug/plate and upwards in the presence of S9 -mix.

After that, Lemonile was tested up to concentrations of 2000 ug/plate in the strains TA1535, TA1537, TA98 and TA102. Severe toxicity was observed in all tester strains.

In additional preincubation assay, Lemonile was tested up to concentrations of 333 ug/plate in the strain TA98 in the presence of S9 -mix.

Lemonile did not induce a dose-related increase in the number of revertant (His*) colonies in each of the five tester strains (TA1535, TA1537, TA98, TA100 and TA102) both in the absence and presence of S9 -metabolic activation. These results were confirmed in separate experiments.

Based on results of this study it is concluded that Lemonile is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003-10-23 to 2004-03-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of inspection - 2011-06-11 to 2011-06-13; Date of signature 2002-01-14
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Name of test material (as cited in study report): Lemonile
- Substance type: Colourless to pale yellow liquid
- Physical state: Liquid
- Analytical purity: 100 % (GC)
- Impurities (identity and concentrations): Not applicable
- Composition of test material, percentage of components: No data
- Isomers composition: No data
- Purity test date: No data
- Batch No: 9000531494
- Expiration date of the batch: 2005-09-25
- Stability under test conditions: No data
- Storage condition of test material: At room temperature (approx. 20° C)
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: Yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Range between 12.5 and 50.0 µg/L without S9 mix, and 206.3 and 825 µg/L with S9 mix. For full details see under "Any other information on materials and methods including tables" below.
Vehicle / solvent:
- Solvent used: Acetone
- Justification for choice of solvent: Chosen due to its solubility properties and its relative nontoxicity to the cell cultures.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Final concentration - 200 μg/mL (1.6 mM) Migrated to IUCLID6: Without metabolic activation only
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Final concentration - 0.7 - 1.0 μg/mL (2.5 - 3.5 μM) Migrated to IUCLID6: With metabolic activation only
Details on test system and experimental conditions:
CELL CULTURES
- Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing, LMP, Technical University Darmstadt, D-64287 Darmstadt, Germany) were stored in liquid nitrogen in the cell bank of RCC Cytotest Cell Research GmbH allowing the repeated use of the same cell culture batch in experiments. As a consequence of screening for mycoplasm contamination and checking for karyotype stability, both before freezing, the parameters of the experiments remain similar because of standardised characteristics of the cells.
- Thawed stock cultures were propagated at 37 °C in 80 cm² plastic flasks (GREINER, D-72632 Frickenhausen, Germany). About 5 x 105 cells per flask were seeded into 15 mL of MEM (Minimal Essential Medium; SEROMED; D-12247 Berlin) supplemented with 10 % foetal calf serum (FCS; PAA Laboratories GmbH, D-35091 Cölbe, Germany). The cells were subcultured twice weekly. The cell cultures were incubated at 37° C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).

METABOLIC ACTIVATION SYSTEM
Preparation of S9
- Phenobarbital/β-Naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from 8 - 12 week old male Wistar HanIbm rats, weight approx. 220 - 320 g (supplied from RCC Ltd; Laboratory Animal Services, CH-4414 Füllinsdorf, Switzerland) induced by applications of 80 mg/kg bw Phenobarbital i.p. (Desitin; D-22335 Hamburg, Germany) and β-Naphthoflavone p.o. (Aldrich, D-89555 Steinheim, Germany) each on 3 consecutive days. The livers were prepared 24 hrs after the last treatment. The S9 fractions were produced by dilution of the liver homogenate with a KCl solution (1:3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in ampoules at -80 °C. Small numbers of the ampoules were kept at -20 °C for up to 1 wk.
- The protein concentration was 34.6 mg/mL (Lot No. 010803) in the pre-experiment, Experiment IA, and Experiment II and 35.6 mg/mL (Lot. No. 160104) in Experiment IB.

Preparation of S9 mix
- An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix to reach the following concentrations: 8 mM MgCl₂, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP, in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
- During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al. (1977).

RANGE-FINDING STUDY
- A pre-test on cell growth inhibition with 4 hrs and 24 hrs treatment was performed in order to determine the toxicity of the test item.
- General experimental conditions were the same as described below for the cytogenetic main experiment.

DOSE SELECTION
- Doses were selected in accordance with the OECD Test Guideline 473.
- With respect to the molecular weight of LEMONILE, 1650 μg/mL (approx. 10 mM) of the test item was applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations between 12.9 and 1650 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity.
- For details on doses applied, see under "Any other information on materials and methods inc. tables" section below.

- Using reduced cell numbers as an indicator for toxicity in the pre-test, clear toxic effects were observed after 4 hrs treatment with 103.1 μg/mL and above in the absence of S9 mix. In addition, 4 hrs treatment with 1650 μg/mL in the presence of S9 mix induced strong toxic effects. Considering the toxicity data of the pre-test, 150 μg/mL (without S9 mix) and 1650 μg/mL (with S9 mix) were chosen as top concentrations in experiment I. Due to slightly increased numbers of endomitotic cells in the test item treated test groups in the presence of S9 mix this part was repeated with concentrations between 100 and 1000 μg/mL, designated experiment IB.

- Dose selection of experiment II was also influenced by test item toxicity. In the range finding experiment clearly reduced cell numbers were observed after 24 hrs exposure with 103.1 μg/mL and above. Therefore, 100 μg/mL were chosen as top treatment concentration for continuous exposure in the absence of S9 mix. Due to missing toxicity and therefore the loss of confirmation of the toxicity data of the pre-experiment, the experimental part with 18 hrs exposure without S9 mix was repeated with a top test item concentration of 200 μg/mL. In the presence of S9 mix 1650 μg/mL were chosen as top treatment concentration with respect to the results obtained in experiment I.

SCHEDULE
- For detailed schedule, please see under "Any other information on materials and methods inc. tables" section below.

SEEDING OF THE CULTURES
Exponentially growing stock cultures more than 50 % confluent were treated with trypsin-EDTA solution at 37 °C for approx. 5 mins. Then the enzymatic treatment was stopped by adding complete culture medium and a single cell suspension is prepared. The trypsin concentration for all subculturing steps is 0.5 % (w/v) in Ca-Mg-free salt solution (Invitrogen GIBCO, D-76131 Karlsruhe, Germany).

TREATMENT
4hr Exposure period:
- The culture medium of exponentially growing cell cultures was replaced with serum-free medium (for treatment with S9 mix) or complete medium (for treatment without S9 mix) with 10 % FCS (v/v), containing the test item. For the treatment with metabolic activation 50 μL S9 mix per mL medium were used. Concurrent negative, solvent, and positive controls were performed.
- After 4 hrs the cultures were washed twice with "Saline G" and then the cells were cultured in complete medium for the remaining culture time.
- The "Saline G" solution was composed as follows (per L): 8000 mg NaCl, 400 mg KCl, 1100 mg glucose, 290 mg Na₂HPO₄.7H₂O, 150 mg KH₂P0₄. pH was adjusted to 7.2

18/28 hr exposure periods:
- The culture medium of exponentially growing cell cultures was replaced with complete medium (with 10 % FCS) containing different concentrations of the test item without S9 mix. The medium was not changed until preparation of the cells.

All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO₂ (98.5 % air).

PREPARATION OF THE CULTURES
- 15.5 hrs and 25.5 hrs, respectively after the start of the treatment colcemid was added (0.2 μg/mL culture medium) to the cultures. 2.5 hrs later, the cells on the slides were treated in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37 °C. After incubation in the hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid (3:1 parts respectively). Per experiment two slides per group were prepared. After preparation the cells were stained with Giemsa (E. Merck, D-64293 Darmstadt, Germany).

DETERMINATION OF CYTOTOXICITY
- For evaluation of cytotoxicity indicated by reduced cell numbers additional 2 cultures per test item and solvent control group, not treated with colcemid, were set up in parallel. These cultures were stained after 18 hrs and 28 hrs, respectively, in order to determine microscopically the cell number within 10 defined fields per coded slide.

ANALYSIS OF METAPHASE CELLS
- Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using Nikon microscopes with 100 × oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells in 500 metaphase cells per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype). Additionally the number of endomitotic cells scored at the evaluation of polyploid cells was noticed and reported (% endomitotic metaphases).
Evaluation criteria:
A test item is classified as non-clastogenic if:
− the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the laboratory's historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
and/or
− no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as clastogenic if:
− the number of induced structural chromosome aberrations is not in the range of the laboratory's historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
and
− either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

Both biological and statistical significance (see below) should be considered together. If the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance should be discussed and/or a confirmatory experiment should have been performed.

Although the inclusion of the structural chromosome aberrations was the purpose of this study, it was important to include the polyploids and endoreduplications. The following criterion was valid:
A test item can be classified as aneugenic if:
− the number of induced numerical aberrations is not in the range of the laboratory's historical control data (0.0 - 8.5 % polyploid cells).
Statistics:
Fisher's exact test. Statistical significance is judged at the standard 5 % level.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE FINDING STUDY
Clear toxic effects were observed after treatment with 103.1 µg/mL and above in the absence of S9 mix and with 1650 µg/mL in the presence of S9 mix. In addition, 24 hours continuous treatment with 103.1 µg/mL and above in the absence of S9 mix induced strong toxic effects (see Table 4 in attached documents).
No precipitation of the test item in culture medium was observed and no relevant influence of the test item on the pH value or osmolarity was observed.

DEFINITIVE STUDY
In both experiments in the absence of S9 mix, no toxic effects indicated by reduced cell numbers or mitotic indices below 50% of the control were observed at the test item concentrations evaluated (see Tables 5, 6 and 10 in attached documents).
In the presence of S9 mix, clearly reduced cell numbers were observed after treatment with 825 µg/mL (42% of control) in Experiment I at the 18 hours preparation interval and with 206.3 µg/mL (46% of control) in Experiment II at the 28 hours preparation interval (see Table 5 in attached documents). No relevant decrease of the mitotic activity was observed (see Tables 6 and 13 in attached documents).
In both cytogenetic experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed (see Tables 8-9 and Tables 15-17 in attached documents). The aberration rates of the cells after treatment with the test item (0.0 - 2.0 % aberrant cells, exclusive gaps) were within the range of the solvent control values (0.5 - 2.0 % aberrant cells, exclusive of gaps) and within the range of the historical control data: 0.0 - 4.0 % aberrant cells, exclusive of gaps. However, in Experiment IB, a single significant increase (3.5%) was observed but this was clearly within the historical control data range and was therefore considered biologically irrelevant.

In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item as compared to the rates of the solvent controls (see Tables 6 and 10 in attached documents).

In experiment IA, after 4 hours treatment in the presence of S9 mix at 18 hours preparation interval the rate of endomitotic metaphases was slightly dose-independently increased (0.8 - 1.5 %) compared with the respective solvent control value (0.2 %) (see Table 7 in attached documents). Generally, no endoreduplications were observed in the control groups. This single observation was not confirmed in one of the other experimental parts performed (see Table 14 in attached documents), regarding that lower test item concentrations were evaluated in these parts and regarding that the test design or schedule was differing. Therefore, a repeat experiment with 4 hours treatment at 18 hours preparation interval with metabolic activation was performed to prove this observation, designated experiment IB. Again the rate of endomitotic metaphases was slightly increased (0.3 - 1.2 %) compared with the respective solvent control value (0.0 %) (see Table 11 in attached documents). The values were not a dose-related increase.

Finally, due to missing dose-dependency in combination with the loss of historical data and due to missing positive findings by scoring the rate of polyploid metaphases and regarding the reason that the main topic of the chromosome aberration test is not the detection of aneugenic effects, this observation was regarded as being of low relevance.

In both experiments, EMS (200 μg/mL) and CPA (0.7 and 1.0 μg/mL, respectively) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations. But, in experiment II in the absence of S9 mix after 28 hours treatment with 200 μg/mL EMS the aberration rate (7.5 %) was slightly lower than the historical positive control data range: 8.0 % - 100.0 % aberrant cells, exclusive gaps. However, the number of cells carrying chromosome aberrations was clearly increased compared with the value of the corresponding negative control (2.0 % aberrant cells, exclusive gaps) and with the laboratory’s historical negative control data: 0.0 - 4.0 % aberrant cells, exclusive of gaps. Therefore, this deviation had no detrimental impact on the validity of this experimental part.

Remarks on result:
other: all strains/cell types tested
Conclusions:
Lemonile did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) with and without metabolic activation when tested up to cytotoxic concentrations, therefore is considered to be non-clastogenic.
Executive summary:

The test item LEMONILE, dissolved in acetone, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in three independent experiments. The following study design was performed:

 

Without S9-mix

 

With S9-mix

 

 

experiment I

experiment II

Exp IA and IB

Exp II

Exposure period

4 hrs

18 hrs

28 hrs

4 hrs

4 hrs

Recovery

14 hrs

-

-

14 hrs

24 hrs

Preparation interval

18 hrs

18 hrs

28 hrs

18 hrs

28 hrs

 

In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations. The highest applied concentration in the pre-test on toxicity (1650 μg/mL; ~10 mM) was chosen with regard to the molecular weight of the test item with respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiments was performed considering the toxicity data. The chosen treatment concentrations are described in Table 2 (page 17). The evaluated experimental points and the results are summarised in Table 1 (page 10 f.). Toxic effects indicated by reduced cell numbers of below 50 % of control were observed in all experiments in the presence of S9 mix. In the absence of S9 mix, no clear cytotoxicity was observed at the concentrations evaluated. However, concentrations showing clearly reduced cell numbers or mitotic indices were not evaluable for cytogenetic damage.

In the three independent experiments, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. However, in experiment IB in the presence of S9 mix a single significant increase (3.5 %) was observed but was within our historical control range (0.0 - 4.0 % aberrant cells, exclusive gaps) and is regarded as being biologically irrelevant. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls.

In experiment IA, after 4 hrs treatment in the presence of S9 mix the number of endoreduplicated metaphases was slightly dose-independent increased (see Table 7, page 28). In the repeat experiment IB this finding was confirmed (see Table 11, page 31), In contrary, in non of the additional experimental parts (see Table 14, page 34) an increase of the number of endoreduplicated metaphases were observed. Due to the missing dose-relation and the low response with a maximum value of 1.5 % endomitotic cells, this observation has to be considered as being biologically irrelevant.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

 

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, LEMONILE is considered to be non-clastogenic in this chromosome aberration test with and without metabolic activation when tested up to cytotoxic concentrations.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Nov2018 - January 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study
Justification for type of information:
Following an ECHA decision CCH-D-2114394631-45-01/F on EC:263-214-5 (3,7-dimethylnona-2,6-dienenitrile), it was requested to conduct additional toxicological studies:
- In vitro gene mutation study in mammalian cells, OECD 476;
- Screening for reproductive/developmental toxicity in rats, oral route, OECD 421,
- Sub-chronic toxicity study (90-day), oral route, in rats, OECD 408,
- Pre-natal developmental toxicity study, oral route, rats or rabbits, OECD 414,
- Identification of degradation products.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
Batch: PE00225096
Purity >=98%
Colorless to pale yellow liquid
Expiry date Nov2020
Target gene:
Hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt)
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
in the presence and absence of an exogenous metabolic activation system, as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr). Dimethyl sulfoxide (DMSO) was used as the vehicle.
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : S9 was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice. The S9 (Lot No. 3998, Expiration Date: 28 Aug 2020) was purchased commercially from Moltox (Boone, NC).
- method of preparation of S9 mix: The S9 mix was prepared on the day of use. Components in the test system:
NADP (sodium salt) (0.8 mM) - Glucose-6-phosphate (1 mM) - Calcium chloride (2 mM) - Potassium chloride (6 mM) - Magnesium chloride (2 mM) - Sodium Phosphate (10 mM) - S9 homogenate (20 μL/mL)
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each lot of S9 was assayed for sterility and its ability to metabolize at least two pro-mutagens to forms mutagenic to Salmonella typhimurium TA100.
Test concentrations with justification for top dose:
Preliminary toxicity assay:
Cells were treated with 10 test substance concentrations, as well as the vehicle control, in the presence and absence of S9 using single cultures. The maximum concentration evaluated was based on the molecular weight of the test substance. The highest concentration tested was 10 mM.

With and without S9 activation: 3.19, 6.38, 12.8, 25.5, 51.0, 102, 204, 408, 816 and 1633 μg/mL

The osmolality of the cultures was acceptable as it did not exceed the osmolality of the vehicle control by more than 120%. The test substance did not have an adverse impact on the pH of the cultures (pH 7.5 at the top dose).
Adjusted relative survival was 5.17 and 21.04% at a concentration of 1633 μg/mL with and without S9, respectively.
The top dose of the definitive test was based upon the results of this preliminary toxicity assay.


Definitive test:
Without activation: 25.5, 51, 102, 204, 408, 816 and 1633 μg/mL
With S9 mix activation: 218, 291, 388, 517, 688, 918 and 1633 μg/mL
Vehicle / solvent:
DMSO
DMSO was choosen as vehicle based on the solubility of the test substance and compatibility with the target cells.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
Preliminary toxicity test: 1 x 10^6 cells in 5 mL; determination of initial survival (after 5 +/- 0.5 hours treatment): 200 cells/60-mm dish in 5 mL

Definitive test:
5 x 10^6 cells in 10 mL
Subculture for phenotypic expression (after 5 hours treatment and each 2-3 days): 2.4 x 10^6 cells/225-cm2 flask in 30 mL; culture for initial survival: 200 cells/60-mm plate in 5 mL

Mutant selection:
6 x 10^5 cells/150-mm plate in 30 mL; cloning efficiency: 200 cells/60-mm plate in 5 mL

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration: 5 +/- 0.5 hours


FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7 days
- Selection time (if incubation with a selective agent): 7 days
- Selective agent: 6-thioguanine (TG) at 10μM, 7 days exposure at the end of the phenotypic expression period
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 2.4 x 10^6 cells from each culture will be plated at a density of 6 x 10^5 cells/150-mm plate (4 plates total) in 30 mL Complete Ham’s F12-Hx containing 10 μM TG.
Three 60-mm plates also were plated, at 200 cells/plate in 5 mL Complete Ham’s F12-Hx in triplicate, to determine the cloning efficiency at the time of selection.
After the 7- day incubation period, the colonies were fixed with methanol, stained with crystal violet and counted. Mutant frequencies were expressed as the number of TGr mutants/10^6 clonable cells. The number of clonable cells was determined from the triplicate 60-mm plates.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method : the cytotoxicity was expressed as the adjusted relative survival (ARS)

Adjusted Relative Survival (ARS) = [(CE (treated) x cell density (treated)) / (CE (control) x cell density (control))] x 100%

Cloning Efficiency (CE) = (number of colonies / number of cells plated) x 100%

METHODS FOR MEASUREMENTS OF GENOTOXICIY

Mutant frequency = (number of mutant colonies / number of cells selected)/CE
Rationale for test conditions:
Cells were treated with 10 test substance concentrations, as well as the vehicle control, in the presence and absence of S9 using single cultures. The maximum concentration evaluated was based on the molecular weight of the test substance. The highest concentration tested was 10 mM. Lower concentrations were prepared by 2-fold dilutions. The pH of the treatment medium was measured, and no pH adjustment was necessary to maintain neutral pH. The osmolality of the solvent control, highest dose level, lowest precipitating dose level and the highest soluble dose level in treatment medium also was measured at the beginning of treatment. Precipitation was assessed at the beginning and end of treatment. Concentrations evaluated in the definitive mutation assay were based on adjusted relative survival, calculated as described below.

Cloning Efficiency* (CE) = (number of colonies / number of cells plated ) x 100%

Adjusted Relative Survival (ARS) = [(CE (treated) x cell density (treated)) / (CE (control) x cell density (control))] x 100%

*CE is equivalent to absolute cloning efficiency
Evaluation criteria:
The test substance was considered to have produced a positive response if it induces a dose-related increase in mutation frequency and an increase exceeding 95% historical vehicle control limits in at least one test dose level(s) as compared with concurrent vehicle control (p<0.01). If only one criterion was met (a statistically significant or dose-dependent increase or an increase exceeding the historical control 95% confidence interval), the result were considered equivocal. If none of these criteria were met, the results were considered to be negative.

Other criteria also may be used in reaching a conclusion about the study results (e.g., comparison to historical control values, biological significance, etc.). In such cases, the Study Director used sound scientific judgment and clearly reported and described any such considerations.
Statistics:
Statistical analyses were performed using the method of Snee and Irr (1981), with significance established at the 0.05 level.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Adjusted relative survival was 5.17 and 21.04% at a concentration of 1633 μg/mL with and without S9, respectively.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under filtered light.
Preliminary Toxicity Test for Selection of Dose Levels:
Positive controls plated concurrently with the mutagenicity assay are listed in the following table. Ethylmethanesulfonate (EMS) was diluted in DMSO (EMS obtained from Acros and DMSO obtained from Sigma-Aldrich) and used as the positive control for the non-activated test system. Benzo(a)pyrene [B(a)P] was also diluted in DMSO (both obtained from Sigma-Aldrich) and used as the positive control for the S9-activated system. Cells were treated with 10 test substance concentrations, as well as the vehicle control, in the presence and absence of S9 using single cultures. The maximum concentration evaluated was based on the molecular weight of the test substance. The highest concentration tested was 10 mM. Lower concentrations were prepared by 2-fold dilutions. The pH of the treatment medium was measured, and no pH adjustment was necessary to maintain neutral pH. The osmolality of the solvent control, highest dose level, lowest precipitating dose level and the highest soluble dose level in treatment medium also was measured at the beginning of treatment. Precipitation was assessed at the beginning and end of treatment. Concentrations evaluated in the definitive mutation assay were based on adjusted relative survival, calculated as described below.

Treatment:
Cells were plated (on Day -1) in 25-cm2 cultures at a density of ~1 x 106 in 5 mL Complete Ham’s F12. Following an overnight incubation under standard conditions, the cultures were washed twice (on Day 0) with Hank’s Balanced Salt Solution (HBSS) and re-fed with 6 mL treatment medium, or 4.8 mL treatment medium plus 1.2 mL S9 mix, as appropriate. Following addition of the test or control substance dose formulations (60 μL) to the flasks, the cultures were incubated under standard conditions for 5 ± 0.5 hours.

Determination of Initial Survival:
After the 5-hour treatment, the treatment media were removed, the cultures were washed twice with 5 mL Ca/Mg-free HBSS (CMF-HBSS), trypsinized and counted once to determine cell density. An aliquot of cells from each culture was plated at a density of 200 cells/60-mm dish in 5 mL Complete Ham’s F12 in triplicate. These plates were incubated for 7 days under standard conditions, and the resulting colonies were fixed with methanol, stained with crystal violet, and counted. The cloning efficiency (%) was calculated for each culture (average of three plates unless any were lost due to technical error), and the cytotoxicity was expressed as the adjusted relative survival (%; relative cloning efficiency x relative cell density at the time of cloning, as compared to the concurrent vehicle control).

CHO/HPRT Mutagenicity Assay:
Seven test substance concentrations, as well as the appropriate positive and vehicle controls, were tested in duplicate cultures with and without S9. The pH of the treatment medium was measured, and no pH adjustment was necessary to maintain neutral pH. Precipitation was assessed at the beginning and end of treatment.
Lemonile (CAS# 61792-11-8) was evaluated at concentrations of 3.19, 6.38, 12.8, 25.5, 51.0, 102, 204, 408, 816 and 1633 μg/mL. The maximum concentration evaluated was based on the molecular weight of the test substance. Highest concentration tested was 10 mM.
The osmolality of the cultures was acceptable as it did not exceed the osmolality of the vehicle control by more than 120%. The test substance did not have an adverse impact on the pH of the cultures (pH 7.5 at the top dose).
Adjusted relative survival was 5.17 and 21.04% at a concentration of 1633 μg/mL with and without S9, respectively.

Based upon the results of the preliminary toxicity assay, the concentrations selected for the definitive mutagenicity assay were as indicated:

Treatment Condition: Non-activated/ Treatment Time:5 ± 0.5 hours/ Concentrations (μg/mL): 25.5, 51, 102, 204, 408, 816 and 1633

Treatment Condition: S9- activate / Treatment Time:5 ± 0.5 hours/ Concentrations (μg/mL): 218, 291, 388, 517, 688, 918 and 1633

The test substance did not have an adverse impact on the pH of the cultures (pH 7.5 at the top dose).

The average adjusted relative survival was 117.01 and 71.37% at a concentration of 1633 μg/mL with and without S9, respectively. Cultures treated at concentrations of 218, 388, 688, 918 and 1633 μg/mL with S9 and 25.5, 51, 408, 816 and 1633 without S9 were chosen for mutant selection. No statistically significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with S9 (p > 0.01). Statistically significant increases in the mutant frequency were observed at two highest concentrations of 816 and 1633 μg/mL without S9 activation. However, these numbers were within our 95% vehicle control limit and the vehicle control for this condition was unusually low. Therefore, these increases are not considered to be biologically relevant. The positive controls induced significant increases in mutant frequency (p < 0.01). All positive and vehicle control values were within acceptable ranges, and all criteria for a valid assay were met.

Visible precipitate was observed as indicated in the following table:

 

 

Treatment Condition

 

Treatment Time

Visible precipitate

At the beginning of treatment period

At the end of treatment period

Non-activated

5 ± 0.5 hours

³408µg/mL

-

S9-activated

5 ± 0.5 hours

³408µg/mL

³816 µg/mL

 

The osmolality of the cultures was measured as follows:

 

Concentration Tested

Concentration (µg/mL)

Osmolality (mmol/kg)

Solvent Control

-

392

Highest

1633

365

Lowest precipitating

408

382

Highest soluble

204

386

Based upon the results of the preliminary toxicity assay, the concentrations selected for the definitive mutagenicity assay were as indicated in the following table:


 

Treatment Condition

 

Treatment Time

 

 

Concentrations (µg/mL)

Non-activated

5 ± 0.5 hours

25.5, 51, 102, 204, 408, 816 and 1633

S9-activated

5 ± 0.5 hours

218, 291, 388, 517, 688, 918 and 1633

 

Visible precipitate was observed as indicated in the following table:

 

 

Treatment Condition

 

Treatment Time

Visible precipitate

At the beginning of treatment period

At the end of treatment period

Non-activated

5 ± 0.5 hours

³102 µg/mL

-

S9-activated

5 ± 0.5 hours

³218 µg/mL

-

 

Conclusions:
Under the conditions of the assay described in this report, Lemonile (CAS# 61792-11-8) was concluded to be negative for the induction of forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro mammalian cell forward gene mutation (CHO/HPRT) assay.
Executive summary:

The test substance, Lemonile (CAS# 61792-11-8), was evaluated for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr). Dimethyl sulfoxide (DMSO) was used as the vehicle.

In the preliminary toxicity assay, the concentrations tested were 3.19, 6.38, 12.8, 25.5, 51.0, 102, 204, 408, 816 and 1633 μg/mL. The maximum concentration evaluated was based on molecular weight of the test substance. The highest concentration tested was 10 mM. Visible precipitate was observed at concentrations ≥408 μg/mL at the beginning of treatment and at concentration ≥816 μg/mL by the end of treatment with S9. Adjusted relative survival was 5.17 and 21.04% at a concentration of 1633 μg/mL with and without S9, respectively. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 218, 291, 388, 517, 688, 918 and 1633 μg/mL with S9 and 25.5, 51, 102, 204, 408, 816 and 1633 μg/mL without S9.

In the definitive mutagenicity assay, visible precipitate was observed at all concentrations with S9 and ≥102 μg/mL without S9 at the beginning of treatment and no visible precipitate was observed at the end of treatment. The average adjusted relative survival was 117.01 and 71.37% at a concentration of 1633 μg/mL with and without S9, respectively. Cultures treated at concentrations of 218, 388, 688, 918 and 1633 μg/mL with S9 and 25.5, 51, 408, 816 and 1633 μg/mL without S9 were chosen for mutant selection. No statistically significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with S9 (p > 0.01). Statistically significant increases in the mutant frequency were observed at two highest concentrations of 816 and 1633 μg/mL without S9 activation. However, these numbers were within our 95% vehicle control limit and the vehicle control for the without activation system was unusually low. Therefore, these increases are not considered to be biologically relevant. The positive controls induced significant increases in mutant frequency (p < 0.01).

These results indicate Lemonile (CAS# 61792-11-8) was negative for the ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro gene mutation study in bacteria:

In a reliable GLP OECD guideline 471 study (NOTOX, 2002), Lemonile was tested in a bacterial reverse mutation assay in Salmonella typhimurium (strains TA98, TA100, TA102, TA1535 and TA1537) with and without metabolic activation (S9). Lemonile did not induce a dose related increase in the number of revertant (His+) colonies in each of the five tester strains both in the absence and presence of S-9 metabolic activation. The test item, Lemonile was therefore considered to be non-mutagenic.

 

In vitro chromosome aberration study in Chinese hamster V79 cells:

In a reliable GLP OECD guideline 473 study (RCC, 2004), Lemonile was tested for its ability to induce structural chromosomal aberrations in V79 cells of the Chinese hamster in vitro in the presence and absence of metabolic activation. The test item did not induce any biologically relevant structural chromosome aberrations. Therefore, Lemonile was considered to be non-clastogenic to Chinese hamster V79 cells in vitro.

 

In vitro gene mutation study in mammalian cells (HPRT):

Lemonile was negative for the ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system.

 

In vivo mutagenicity study in mammalian cells:

In a reliable GLP OECD guideline 474 study (RCC, 2005), the potential of Lemonile to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse was investigated. The test item did not induce micronuclei and therefore Lemonile is considered to be non-mutagenic.


Short description of key information:
Ames test: negative (OECD 471)
Chromosome aberration test: negative (OECD 473)
Mouse micronucleus test: negative (OECD 474)

Mammalian Cell Gene Mutation, HPRT (OECD 476): Negative

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The results of an vitro gene mutation study in bacteria, an in vitro chromosome aberration study in Chinese hamster V79 cells, in vitro mammalian cell gene mutation test and an in vivo mutagenicity study in mammalian cells were all negative. Therefore, it is concluded that Lemonile is not genotoxic and does not warrant classification for mutagenicity according to CLP Regulation EC 1272/2008.